Human respiratory syncytial virus (RSV) is an enveloped RNA-containing virus of Family Paramyxoviridae, Order Mononegavirales, the nonsegmented negative strand RNA viruses. RSV is the most important viral agent of pediatric respiratory tract disease worldwide but lacks an approved vaccine or effective antiviral therapy. Other mononegaviruses include measles, mumps, rabies and Ebola viruses, and hence study of the group is of interest in general. We previously showed that the RSV genome is a single negative strand of RNA of 15,222 nucleotides that encodes 10 major mRNAs and 11 proteins. It thus is one of the more complex mononegaviruses. The purpose of this project is to identify the functions of the viral proteins and to reconstruct events in the viral growth cycle under conditions where they can be more readily studied. Part of these studies involves a minireplicon system, in which a short analog of the genome or its antigenome replicative intermediate is expressed intracellularly from a transfected plasmid and complemented by proteins supplied from other plasmids. Another approach involves infectious recombinant virus bearing mutations in relevant genes. One of the RSV proteins, the M2-1 protein, was identified as a novel transcription antitermination protein. Another protein, M2-2, which is expressed from a separate open reading frame from the same mRNA was identified as a novel RNA regulatory protein. The G glycoprotein had previously been identified as the major attachment protein. However, we were able to delete the G gene from infectious recombinant virus: the resulting delta-G virus replicated as efficiently as wild-type on Vero cells, but was 100- to 1000-fold reduced on HEp-2 cells. Another recombinant that lacked both the G and SH glycoproteins was equally viable, implying that the remaining glycoprotein, F, was sufficient to mediate viral attachment and entry in vitro. However, the delta-G virus was strongly attenuated in mice, indicating that G is very important in vivo. This provides a system in which G mutants can be readily recovered and propagated (in Vero cells) and the functionality of G readily tested (in HEp-2 cells and mice). We also could conveniently test the ability of G to bind to cell surface glycosamino glycans (GAGs), which appear to function as a "G receptor". Using this system, we are performing structure-function studies to test the importance of various G structural features for infectivity.